Alzheimer""s disease is a degenerative brain disorder that is characterized clinically by progressive loss of memory and cognitive impairment. Pathologically, the disease is characterized by lesions comprising neurofibrillary tangles, cerebrovascular amyloid deposits, and neuritic plaques. The cerebrovascular amyloid deposits and neuritic plaques contain amyloid-xcex2 peptide. The aggregation of amyloid-xcex2 peptide is instrumental in the pathogenesis of Alzheimer""s disease.
Amyloid-xcex2 peptide is derived from amyloid-xcex2 precursor protein (APP). APP is a cell-surface protein with a large N-terminal extracellular sequence, a single transmembrane region (TMR) and a short C-terminal cytoplasmic tail. APP is processed by proteolysis in all cells. Initially, xcex1- and xcex2-secretases cleave APP at defined extracellular sequences just outside of the TMR to release a large N-terminal extracellular fragment. Thereafter, xcex3-secretase cuts APP in the middle of the TMR to generate small extracellular peptides and a C-terminal fragment comprising half of the TMR and the full cytoplasmic tail. See, e.g., Selkoe (1998) Trends Cell Biol. 8, 447-453; Bayer et al. (1999) Mol. Psychiatry 4, 524; Haass et al. (1999) Science 286, 916-919; Price et al. (1998) Ann. Rev. Gevet 32, 461-493.
Cleavage of APP by xcex2- and xcex3-secretase produces the amyloid-xcex2 peptides (AP40 and AP42) implicated in the pathogeneses of Alzheimer""s disease. Various methods for the diagnosis and monitoring of the disease involve assessing the cleavage of APP and detection of amyloid-xcex2 peptide. However, these methods suffer from various disadvantages including the insolubility of amyloid-xcex2 peptide, cross-reactivity of antibodies against the peptide with the precursor APP, and different levels of protease activity in different body fluids.
The xcex3-cleavage of APP is mediated by presenilins, intrinsic membrane proteins that may correspond to xcex3-secretase and that are mutated in some cases of familial Alzheimer""s disease. See, e.g., Esler et al. (2000) Nat. Cel. Biol. 2, 428-434. Also, xcex3-cleavage occurs in APP-homologs that are not implicated in Alzheimer""s disease. For example, Notch proteins are membrane proteins that are also cleaved in the middle of the TMR in a presenilin-dependent reaction. See, e.g. Ye et al. (1999) Nature 398, 525-529; De Strooper et al. (1999) Nature 398, 518-522; Struhl et al. (1999) Nature 398, 522-525. Notch proteins are cell-surface proteins involved in intercellular signaling in which presenilin-dependent cleavage liberates a cytoplasmic fragment that functions in nuclear transcription. Struhl et al. (2000) Mol. Cell 6, 625-636. Sterol regulatory element binding proteins (SREPPs) are also cleaved in the TMRs to generate nuclear transcription factors. Brown et al. (2000) Cell 100, 391-398. In contrast, the physiological significance of xcex3-cleavage of APP, and in particular the biological role of the cytoplasmic tail fragment, has heretofore been unclear. In accordance with the present invention it has been discovered that the cytoplasmic tail forms a functional complex with nuclear proteins, and that the complex is a potent stimulator of transcription. The present invention thus provides new methods for identifying agents that affect the cleavage of APP.
The present invention provides a method of identifying an agent that affects the cleavage of APP comprising contacting a cell containing APP modified in the C-terminal cytoplasmic tail to allow detection of nuclear localization with a candidate agent and measuring nuclear localization of a C-terminal cytoplasmic cleavage product of APP in the presence and absence of the agent.
In another embodiment, the present invention provides a method of identifying an agent that affects the cleavage of APP comprising providing a cell containing APP and a protein that interacts with the C-terminal cytoplasmic cleavage product of APP to regulate transcription, wherein the protein is modified to allow detection of nuclear localization of the C-terminal cytoplasmic cleavage product of APP; contacting the cell with a candidate agent; and measuring nuclear localization of the C-terminal cytoplasmic cleavage product of APP in the presence and absence of the agent.
The invention further provides agents identified by the foregoing method, and compositions comprising the agents.
In another embodiment, the invention is directed to vectors, transfected cells and kits useful for identifying an agent that affects the cleavage of APP.